Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Targeting the Mycobacterium tuberculosis transpeptidase LdtMt2 with cysteine-reactive inhibitors including ebselen.

ABSTRACT: The l,d-transpeptidases (Ldts) are promising antibiotic targets for treating tuberculosis. We report screening of cysteine-reactive inhibitors against LdtMt2 from Mycobacterium tuberculosis. Structural studies on LdtMt2 with potent inhibitor ebselen reveal opening of the benzisoselenazolone ring by a nucleophilic cysteine, forming a complex involving extensive hydrophobic interactions with a substrate-binding loop.

SUBMITTER: de Munnik M

PROVIDER: S-EPMC6984337 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Targeting the Mycobacterium tuberculosis transpeptidase Ldt<sub>Mt2</sub> with cysteine-reactive inhibitors including ebselen.

de Munnik Mariska M Lohans Christopher T CT Lang Pauline A PA Langley Gareth W GW Malla Tika R TR Tumber Anthony A Schofield Christopher J CJ Brem Jürgen J

Chemical communications (Cambridge, England) 20190801 69

The l,d-transpeptidases (Ldts) are promising antibiotic targets for treating tuberculosis. We report screening of cysteine-reactive inhibitors against LdtMt2 from Mycobacterium tuberculosis. Structural studies on LdtMt2 with potent inhibitor ebselen reveal opening of the benzisoselenazolone ring by a nucleophilic cysteine, forming a complex involving extensive hydrophobic interactions with a substrate-binding loop. ...[more]

PMID: 31380528

Similar Datasets

A Fluorescence-Based Assay for Screening ?-Lactams Targeting the Mycobacterium tuberculosis Transpeptidase LdtMt2.

Project description:Mycobacterium tuberculosis l,d-transpeptidases (Ldts), which are involved in cell-wall biosynthesis, have emerged as promising targets for the treatment of tuberculosis. However, an efficient method for testing inhibition of these enzymes is not currently available. We present a fluorescence-based assay for LdtMt2 , which is suitable for high-throughput screening. Two fluorogenic probes were identified that release a fluorophore upon reaction with LdtMt2 , thus making it possible to assess the availability of the catalytic site in the presence of inhibitors. The assay was applied to a panel of ?-lactam antibiotics and related inhibitors; the results validate observations that the (carba)penem subclass of ?-lactams are more potent Ldt inhibitors than other ?-lactam classes, though unexpected variations in potency were observed. The method will enable systematic structure-activity relationship studies on Ldts, thereby facilitating the identification of new antibiotics active against M.?tuberculosis.

| S-EPMC7028133 | biostudies-literature

Structure of LdtMt2, an L,D-transpeptidase from Mycobacterium tuberculosis.

Project description:The transpeptidase LtdMt2 catalyzes the formation of the (3-3) cross-links characteristic of the peptidoglycan layer in the Mycobacterium tuberculosis cell wall. Bioinformatics analysis suggests that the extramembrane part of the enzyme consists of three domains: two smaller domains (denoted as A and B domains) and a transpeptidase domain (the C domain) at the C-terminus. The crystal structures of two fragments comprising the AB domains and the BC domains have been determined. The structure of the BC module, which was determined to 1.86 Å resolution using Se-SAD phasing, consists of the B domain with an immunoglobulin-related fold and the catalytic domain belonging to the ErfK/YbiS/YbnG fold family. The structure of the AB-domain fragment, which was solved by molecular replacement to 1.45 Å resolution, reveals that despite a lack of overall sequence identity the A domain is structurally very similar to the B domain. Combining the structures of the two fragments provides a view of the complete three-domain extramembrane part of LdtMt2 and shows that the protein extends at least 80-100 Å from the plasma membrane into the peptidoglycan layer and thus defines the maximal distance at which cross-links are formed by this enzyme. The LdtMt-related transpeptidases contain one or two immunoglobulin domains, which suggests that these might serve as extender units to position the catalytic domain at an appropriate distance from the membrane in the peptidoglycan layer.

| S-EPMC3605044 | biostudies-literature

The Mycobacterium tuberculosis protein LdtMt2 is a nonclassical transpeptidase required for virulence and resistance to amoxicillin.

Project description:The peptidoglycan layer is a vital component of the bacterial cell wall. The existing paradigm describes the peptidoglycan network as a static structure that is cross-linked predominantly by 4-->3 transpeptide linkages. However, the nonclassical 3-->3 linkages predominate the transpeptide networking of the peptidoglycan layer of nonreplicating Mycobacterium tuberculosis. The molecular basis of these linkages and their role in the physiology of the peptidoglycan layer, virulence and susceptibility of M. tuberculosis to drugs remain undefined. Here we identify MT2594 as an L,D-transpeptidase that generates 3-->3 linkages in M. tuberculosis. We show that the loss of this protein leads to altered colony morphology, loss of virulence and increased susceptibility to amoxicillin-clavulanate during the chronic phase of infection. This suggests that 3-->3 cross-linking is vital to the physiology of the peptidoglycan layer. Although a functional homolog exists, expression of ldtMt2 is dominant throughout the growth phases of M. tuberculosis. 4-->3 transpeptide linkages are targeted by one of the most widely used classes of antibacterial drugs in human clinical use today, beta-lactams. Recently, meropenem-clavulanate was shown to be effective against drug-resistant M. tuberculosis. Our study suggests that a combination of L,D-transpeptidase and beta-lactamase inhibitors could effectively target persisting bacilli during the chronic phase of tuberculosis.

| S-EPMC2851841 | biostudies-literature

Structural insight into the inactivation of Mycobacterium tuberculosis non-classical transpeptidase LdtMt2 by biapenem and tebipenem.

Project description:The carbapenem subclass of ?-lactams is among the most potent antibiotics available today. Emerging evidence shows that, unlike other subclasses of ?-lactams, carbapenems bind to and inhibit non-classical transpeptidases (L,D-transpeptidases) that generate 3 ? 3 linkages in bacterial peptidoglycan. The carbapenems biapenem and tebipenem exhibit therapeutically valuable potencies against Mycobacterium tuberculosis (Mtb).Here, we report the X-ray crystal structures of Mtb L,D-transpeptidase-2 (LdtMt2) complexed with biapenem or tebipenem. Despite significant variations in carbapenem sulfur side chains, biapenem and tebipenem ultimately form an identical adduct that docks to the outer cavity of LdtMt2. We propose that this common adduct is an enzyme catalyzed decomposition of the carbapenem adduct by a mechanism similar to S-conjugate elimination by ?-lyases.The results presented here demonstrate biapenem and tebipenem bind to the outer cavity of LdtMt2, covalently inactivate the enzyme, and subsequently degrade via an S-conjugate elimination mechanism. We discuss structure based drug design based on the findings and propose that the S-conjugate elimination can be leveraged to design novel agents to deliver and locally release antimicrobial factors to act synergistically with the carbapenem carrier.

| S-EPMC5445500 | biostudies-literature

The cysteine-reactive small molecule ebselen facilitates effective SOD1 maturation.

Project description:Superoxide dismutase-1 (SOD1) mutants, including those with unaltered enzymatic activity, are known to cause amyotrophic lateral sclerosis (ALS). Several destabilizing factors contribute to pathogenicity including a reduced ability to complete the normal maturation process which comprises folding, metal cofactor acquisition, intra-subunit disulphide bond formation and dimerization. Immature SOD1 forms toxic oligomers and characteristic large insoluble aggregates within motor system cells. Here we report that the cysteine-reactive molecule ebselen efficiently confers the SOD1 intra-subunit disulphide and directs correct SOD1 folding, depopulating the globally unfolded precursor associated with aggregation and toxicity. Assisted formation of the unusual SOD1 cytosolic disulphide bond could have potential therapeutic applications. In less reducing environments, ebselen forms a selenylsulphide with Cys111 and restores the monomer-dimer equilibrium of A4V SOD1 to wild-type. Ebselen is therefore a potent bifunctional pharmacological chaperone for SOD1 that combines properties of the SOD1 chaperone hCCS and the recently licenced antioxidant drug, edaravone.

| S-EPMC5923229 | biostudies-literature

Targeting the cell wall of Mycobacterium tuberculosis: structure and mechanism of L,D-transpeptidase 2.

Project description:With multidrug-resistant cases of tuberculosis increasing globally, better antibiotic drugs and novel drug targets are becoming an urgent need. Traditional ?-lactam antibiotics that inhibit D,D-transpeptidases are not effective against mycobacteria, in part because mycobacteria rely mostly on L,D-transpeptidases for biosynthesis and maintenance of their peptidoglycan layer. This reliance plays a major role in drug resistance and persistence of Mycobacterium tuberculosis (Mtb) infections. The crystal structure at 1.7 Å resolution of the Mtb L,D-transpeptidase Ldt(Mt2) containing a bound peptidoglycan fragment, reported here, provides information about catalytic site organization as well as substrate recognition by the enzyme. Based on our structural, kinetic, and calorimetric data, we propose a catalytic mechanism for Ldt(Mt2) in which both acyl-acceptor and acyl-donor substrates reach the catalytic site from the same, rather than different, entrances. Together, this information provides vital insights to facilitate development of drugs targeting this validated yet unexploited enzyme.

| S-EPMC3573878 | biostudies-literature

Mechanism of inhibition of Mycobacterium tuberculosis antigen 85 by ebselen.

Project description:The increasing prevalence of drug-resistant tuberculosis highlights the need for identifying new antitubercular drugs that can treat these infections. The antigen 85 (Ag85) complex has emerged as an intriguing mycobacterial drug target due to its central role in synthesizing major components of the inner and outer leaflets of the mycobacterial outer membrane. Here we identify ebselen (EBS) as a potent inhibitor of the Mycobacterium tuberculosis Ag85 complex. Mass spectrometry data show that EBS binds covalently to a cysteine residue (C209) located near the Ag85C active site. The crystal structure of Ag85C in the presence of EBS shows that C209 modification restructures the active site, thereby disrupting the hydrogen-bonded network within the active site that is essential for enzymatic activity. C209 mutations display marked decreases in enzymatic activity. These data suggest that compounds using this mechanism of action will strongly inhibit the Ag85 complex and minimize the selection of drug resistance.

| S-EPMC4049535 | biostudies-literature

Targeting Mycobacterium tuberculosis topoisomerase I by small-molecule inhibitors.

Project description:We describe inhibition of Mycobacterium tuberculosis topoisomerase I (MttopoI), an essential mycobacterial enzyme, by two related compounds, imipramine and norclomipramine, of which imipramine is clinically used as an antidepressant. These molecules showed growth inhibition of both Mycobacterium smegmatis and M. tuberculosis cells. The mechanism of action of these two molecules was investigated by analyzing the individual steps of the topoisomerase I (topoI) reaction cycle. The compounds stimulated cleavage, thereby perturbing the cleavage-religation equilibrium. Consequently, these molecules inhibited the growth of the cells overexpressing topoI at a low MIC. Docking of the molecules on the MttopoI model suggested that they bind near the metal binding site of the enzyme. The DNA relaxation activity of the metal binding mutants harboring mutations in the DxDxE motif was differentially affected by the molecules, suggesting that the metal coordinating residues contribute to the interaction of the enzyme with the drug. Taken together, the results highlight the potential of these small molecules, which poison the M. tuberculosis and M. smegmatis topoisomerase I, as leads for the development of improved molecules to combat mycobacterial infections. Moreover, targeting metal coordination in topoisomerases might be a general strategy to develop new lead molecules.

| S-EPMC4325804 | biostudies-literature

Isolation, Purification and Characterization of L,D-transpeptidase 2 from Mycobacterium tuberculosis.

Project description:L,D-transpeptidase 2 from Mycobacterium tuberculosis plays a key role in the formation of nonclassical 3-3 peptidoglycan cross-links in a pathogen's cell wall making it resistant to a broad range of penicillin antibiotics. The conditions of cultivation, isolation, and purification of recombinant L,D-transpeptidase 2 from M. tuberculosis have been optimized in this study. Oxidation of the free SH groups of catalytic cysteine Cys354 is an important factor causing the inactivation of the enzyme, which occurs during both the expression and storage of enzyme preparations. The biochemical characteristics of purified L,D-transpeptidase 2 and L,D-transpeptidase 2 lacking domain A were determined; the kinetic constants of enzyme-catalyzed nitrocefin transformation were evaluated.

| S-EPMC6475871 | biostudies-literature

Selective Covalent Targeting of Anti-Apoptotic BFL-1 by Cysteine-Reactive Stapled Peptide Inhibitors.

Project description:Anti-apoptotic BCL-2 family proteins block cell death by trapping the critical ?-helical BH3 domains of pro-apoptotic members in a surface groove. Cancer cells hijack this survival mechanism by overexpressing a spectrum of anti-apoptotic members, mounting formidable apoptotic blockades that resist chemotherapeutic treatment. Drugging the BH3-binding pockets of anti-apoptotic proteins has become a highest-priority goal, fueled by the clinical success of ABT-199, a selective BCL-2 inhibitor, in reactivating apoptosis in BCL-2-dependent cancers. BFL-1 is a BCL-2 homolog implicated in melanoma, lymphoma, and other cancers, and remains undrugged. A natural juxtaposition of two unique cysteines at the binding interface of the NOXA BH3 helix and BFL-1 pocket informed the development of stapled BH3 peptides bearing acrylamide warheads to irreversibly inhibit BFL-1 by covalent targeting. Given the frequent proximity of native cysteines to regulatory binding surfaces, covalent stapled peptide inhibitors provide a new therapeutic strategy for targeting pathologic protein interactions.

| S-EPMC5055752 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data